The present invention relates generally to nucleic acids and polypeptides characteristic of receptors for mammalian interleukin-10, and more particularly to their uses in preparing reagents useful for diagnosing or treating various receptor-related medical conditions.
Activated hemopoietic cells secrete numerous proteins. Cytokines are a subset of these proteins and play a variety of important roles in regulation of immune responses by controlling proliferation, differentiation, and the effector functions of immune cells. Most cytokines have more than one biological activity and which activity is the most important likely depends on the local context in which the cytokine is produced.
These cytokines are intercellular signaling molecules whose actions are typically mediated through specific receptor molecules found on target cells. The structure and mechanism of action of these receptors on target cells is not well understood, though many are composed of at least two separate proteins. These earlier described heterodimeric receptors are often composed of polypeptides which are related both to each other and to receptors for other cytokines. Components of receptors for other cytokines have been described. See, e.g., Gearing, et al. (1989) EMBO J. 8:3667-3676 (low affinity xcex1 chain of a human GM-CSF receptor); Itoh, et al. (1990) Science 247:324-327 (low affinity xcex1 chain of a mouse IL-3 receptor); and Hayashida, et al. (1990) Proc. Nat""l Acad. Sci. USA 87:9655-9659 (a xcex2 chain of a human GM-CSF receptor); and Tavernier, et al. (1991) Cell 66:1175-1184 (IL-5 receptor, xcex1 and xcex2 chains). The various components of the earlier identified receptors appear to share properties useful in defining a receptor superfamily of related proteins. See, e.g., Bazan (1990) Immunology Today 11:350-354; and Bazan (1990) Proc. Nat""l Acad. Sci. USA 87:6934-6938. However, the structure and mechanism of action of a receptor for a mammalian interleukin-10 (IL-10) could not be predicted with reliability based merely upon speculated similarity to receptors for other cytokines.
As soluble intercellular messenger molecules, the cytokines likely bind to cellular receptors, e.g., cell surface receptors. Receptor molecules have been identified and isolated for G-CSF, GM-CSF, EPO, TNF, IFN-xcex3, IL-2, IL-3, IL-4, IL-5, IL-6, and IL-7. Many of these receptors have two chains, both of which are members of the hemopoietic receptor superfamily. In such cases, typically one chain, designated the xcex1 chain, can bind its ligand with low affinity. This interaction may or may not result in transduction to the cell of a signal. Another chain, designated the xcex2 chain, when associated with the xcex1 chain, confers higher affinity binding of the heterodimeric receptor to the cytokine. The xcex2 chain by itself usually lacks significant ligand binding affinity. The dimeric form of receptor is capable of transducing a signal into the cell as a consequence of ligand, e.g., cytokine, binding. However, any similarity between the structural and functional features of those receptors generally and a receptor for IL-10 is speculative. Additional subunits may also be associated with the receptors.
A cytokine synthesis inhibitory factor (CSIF) activity led to assays which allowed the isolation of a cytokine designated interleukin-10 (IL-10). See Fiorentino, et al. (1989) J. Exptl. Med. 170:2081-2095; and Mosmann, et al. (1991) Immunol. Today 12:A49-A53. Both mouse and human counterparts have been isolated. See Moore, et al. (1990) Science 248:1230-1234; and Vieira, et al. (1991) Proc. Nat""l Acad. Sci. USA 88:1172-1176. A human viral analog, known as either vIL-10 or BCRF1, has been described which shares many characteristic activities of the natural human form. See Hsu, et al. (1990) Science 250:830-832. Another viral homolog has been described from an equine herpes virus. See Rode, et al. (1993) Virus Genes 7:111-116.
Human IL-10 (hIL-10) has an N-terminal hydrophobic signal sequence of 18 amino acids, one potential N-linked glycosylation site, 4 cysteine residues, and seven methionine residues. It shares strong DNA and amino acid sequence homology with mouse IL-10 and an open reading frame in Eppstein-Barr virus, BCRF1, and an open reading frame in an equine herpes virus, type II. It inhibits cytokine synthesis by activated T cells, stimulates growth for thymocytes and mast cells, induces class II MHC expression, and sustains viability in culture of small dense resting mouse B cells. A mouse counterpart has also been described, and equivalent proteins would be found in other mammalian species. IL-10 binding to cell surface receptors is thought to be an initiating step for various specific cellular responses, as described below.
One means to modulate IL-10 effect upon binding to its receptor, and therefore potentially useful in treating inappropriate immune responses, e.g., autoimmune, inflammation, sepsis, and cancer situations, is to inhibit the receptor signal transduction. Unfortunately, finding reagents capable of serving as an antagonist or agonist has been severely hampered by the absence of large quantities of IL-10 receptor, preferably purified and in an active form. In order to characterize the structural properties of the IL-10 receptor in greater detail and to understand the mechanism of action at the molecular level, purified receptor will be very useful.
Moreover, similarities to other cytokine functions exist. In particular, the IL-10 receptor likely shares many functions and characteristics with other receptors, but also exhibits different structural and functional properties. The receptors provided herein, by comparison to other receptors or by combining structural components, will provide further understanding of signal transduction induced by ligand binding.
The isolated receptor gene should provide means to generate an economical source of the receptor, allow expression of more receptors on a cell leading to increased assay sensitivity, promote characterization of various receptor subtypes and variants, and allow correlation of activity with receptor structures. Moreover, fragments of the receptor may be useful as agonists or antagonists of ligand binding. See, e.g., Harada, et al. (1992) J. Biol. Chem. 267:22752-22758.
Thus, a need exists for the isolation and characterization of nucleic acids encoding components of receptors for IL-10. The present invention provides these and the means for preparing many other useful reagents.
The present invention provides nucleic acid and protein sequences of components of a receptor for IL-10. Both a human IL-10 receptor component and a mouse counterpart are exemplified, though equivalent components from other mammalian species will be found by similar methods or based upon other properties derived therefrom.
The present invention provides recombinant or isolated nucleic acids comprising a sequence exhibiting homology to a sequence encoding a mammalian receptor for IL-10, a fragment thereof, or a unique portion thereof. In preferred embodiments, the nucleic acids will comprise deoxyribonucleic acid, will be isolated, further comprise a regulatory sequence from the 5xe2x80x2 or 3xe2x80x2 sequence adjacent a gene encoding a receptor for IL-10, or are operably linked to a genetic control element. In alternative embodiments the receptors, fragments, or portions thereof have a biological activity, e.g., one characteristic of a receptor for IL-10, or are from a mammal, including a mouse or human.
In particular embodiments, the nucleic acids, are capable of hybridizing at high stringency to SEQ ID NO: 1 or 3, or are isolated using a probe which hybridizes at high stringency to a human receptor for IL-10. The invention also embraces nucleic acids capable of hybridizing to these sequences which contain mutations selected from the group consisting of nucleotide substitutions, nucleotide deletions, nucleotide insertions, and inversions of nucleotide stretches. Alternative embodiments include recombinant nucleic acids which are operably linked to a genetic control element, e.g., a prokaryotic promoter element or a eukaryotic expression control element, including a viral promoter.
Various embodiments include expression vectors for expressing DNA encoding a receptor for IL-10, or vectors comprising these sequences and a selection marker. The invention also embraces host cells comprising an expression vector which is capable of expressing these receptors. Preferred host cell embodiments include prokaryotes, including gram negative and gram positive bacteria, including E coli; lower eukaryotes, including yeasts; and higher eukaryotes, including animal cells, including mammalian cells, including human. Preferably the receptor is selected from a human receptor for IL-10; or a mouse receptor for IL-10. Other embodiments include nucleic acids further encoding a second protein or polypeptide, e.g., where the second polypeptide is fused to the receptor. The invention further embraces subcellular structures, cells, or organisms comprising these nucleic acids.
The present invention also embraces proteins or polypeptides encoded by these DNA sequences, preferably which are substantially free of protein or cellular contaminants, other than those derived from a recombinant host. The receptor proteins or polypeptides will often be from a mammal, including a mouse or human, and can have an amino acid sequence as found in SEQ ID NO: 2 or 4, or an allelic or species variant thereof, or a unique portion thereof. The receptor proteins or polypeptides can be attached to a solid support, be substantially pure, or be in a pharmaceutically acceptable form, with or without additional carriers or excipients. The invention also conceives of fusion proteins or polypeptides, including those further comprising a sequence from a second receptor protein. Other embodiments include subcellular structures, cells, or organisms comprising such receptor proteins or polypeptides.
The invention also provides methods for producing receptor proteins or polypeptides comprising culturing a cell comprising a described nucleic acid in a nutrient medium; and expressing the receptor proteins or polypeptides in the cell. Various alternative embodiments further comprise a step of purifying the receptor proteins or polypeptides, where the receptor proteins or polypeptides are secreted into the medium and purified therefrom, and wherein the receptor is from a mammal, including a mouse or human. The invention also provides receptors made by these methods and exhibiting a post-translational modification pattern distinct from that in normal native receptor, e.g., glycosylation; alkylation; and carboxylation. The receptor can be made in a cell line expressing a receptor exhibiting a non-natural receptor glycosylation pattern. The invention also provides methods for diagnosing a medical condition characterized by inappropriate IL-10 response in a host comprising contacting a sample from the host with a specific binding reagent to a nucleic acid encoding a receptor for IL-10 or fragment thereof; or to a receptor for IL-10 or fragment thereof, and measuring the level of binding of the reagent to the sample. In various alternatives, the binding reagent is a nucleic acid probe for a gene encoding the receptor or fragment thereof, an antibody which recognizes a receptor for IL-10 or a fragment thereof; or a ligand, agonist, or antagonist for a receptor for IL-10. Preferably the receptor is from a mammal, including a mouse or human.
The invention also provides methods of screening for a compound having binding affinity to a receptor for IL-10, comprising producing an isolated or recombinant receptor by a method as described; and assaying for the binding of the compound to the receptor, thereby identifying compounds having defined binding affinity therefor. Preferably, the compound is a ligand, agonist, or antagonist for these receptors.
The present invention also provides proteins and polypeptides, e.g., free of proteins with which they are naturally associated and having an amino acid sequence homologous to a fragment of a receptor for IL-10. Typically, the receptor is from a mammal, including a mouse or human, and specific embodiments have sequence of SEQ ID NO: 2 or 4.
The invention encompasses a recombinant or substantially pure polypeptides comprising a region exhibiting substantial identity to an amino acid sequence of a receptor for IL-10. Particular embodiments include polypeptides having a sequence selected from SEQ ID NO: 2 or 4, or polypeptides attached to a solid support.
The present invention provides various antibodies having binding affinity to a recombinant receptor for IL-10, or a fragment thereof. Preferred embodiments are raised against the receptor for IL-10, and can be either neutralizing or non-neutralizing antibodies fused to a toxic moiety, or conjugated to a marker moiety, including a radionuclide. Preferably, the antibody binds to a receptor from a mammal, including a mouse or human.
Additionally, the invention provides methods of treating a host having a medical condition characterized by inappropriate IL-10 response or exhibiting abnormal expression of a receptor for IL-10, comprising administering to the host a therapeutically effective amount of a composition comprising (a) an antibody which binds to a receptor for IL-10 or fragment thereof; (b) a ligand, agonist, or antagonist for a receptor for IL-10; or (c) a ligand binding receptor, or fragment thereof, for IL-10. In one embodiment, the antibody is a monoclonal antibody. In others, the agonist or antagonist is selected by a method of contacting a target compound with (a) isolated or recombinant receptor for IL-10, or (b) ligand binding fragment of the receptor; and identifying the target compound with isolated or recombinant receptor for IL-10, or ligand binding fragment of the receptor; and identifying the target compound based upon the effects of the contacting.
The invention also provides methods of evaluating binding affinity of a test compound to a receptor for IL-10, the method comprising contacting (a) a sample containing the receptor, or a fragment thereof, with a labeled compound having known affinity for the receptor; and (b) the test compound; and measuring the level of bound labeled compound, the amount being inversely proportional to the amount of test compound which bound to the receptor. Preferably, the receptor is from a mammal, including a mouse or human. An alternative embodiment is a method of modulating biological activity of a receptor for IL-10, comprising contacting the receptor with a composition selected from an antibody which binds to the receptor; a ligand, agonist, or antagonist for a receptor for IL-10; and a ligand binding fragment from a receptor for IL-10.
The invention also provides useful reagents in kit form. For example, it provides a kit useful for (a) quantifying a receptor for IL-10; or (b) for determining the bindings affinity of a test sample to a receptor for IL-10; the kit comprising a labeled compound having binding affinity for the receptor, and a means for measuring bound labeled compound. Various embodiments include kits further comprising recombinant receptor, wherein the labeled compound is a ligand for the receptor, including IL-10; wherein the compound is an antibody; wherein the means for measuring is a solid phase for immobilizing the receptor; or wherein the solid phase contains a capture molecule. The invention also provides a kit for assaying, in a sample, antibody against a receptor for IL-10, comprising the receptor and an antibody detection means. In one embodiment the receptor is attached to a solid support.
The invention also provides compounds known to modulate activity of a receptor for IL-10, selected by a method of: contacting the compound with isolated or recombinant receptor, or a fragment thereof, for IL-10; and evaluating the effect on biological activity by the contacting.
The invention also provides methods of modulating a biological effect of IL-10, comprising a step of interfering with biological mechanisms, e.g., signal transduction, of a class 2 cytokine receptor, e.g., an interferon receptor. It also provides methods of modulating a biological effect of a class 2 receptor, e.g., an interferon, comprising a step of interfering with biological mechanisms of an IL-10 receptor.
(Numbered references following are found at the end of Example 14)
Using epitope-tagged hIL-10, we identified a cell line expressing hIL-10R and isolated cDNA clones encoding hIL-10R. hIL-10R has 70% and 60% sequence identity to mIL-10R at the nucleic acid and protein levels, respectively (23). COS7 and Ba/F3 cells transfected with hIL-10R cDNA clones express cell-surface receptors which bind hIL-10 (but not mIL-10) specifically with high affinity, and Ba/F3 cells expressing recombinant hIL-10R are stimulated by hIL-10 but not mIL-10. (FIGS. 22A-D)Recombinant hIL-10R is indistinguishable in size from hIL-10R expressed by the cells from which the cDNA was isolated, and moreover, is similar in size to mIL-10R (FIG. 21). hIL-10R mRNA was present in all cells examined which are known to respond to hIL-10 (FIGS. 23A-C). We thus conclude that the hIL-10R identified is a functional receptor for hIL-10, because it binds ligand specifically, mediates transduction of a biological response to hIL-10, and exhibits the known species specificity of the human receptor (1, 46).
We have observed significant levels of hIL-10R mRNA expression by cells about which relatively little is known concerning their-direct. responses to IL-10: T cells and NK/LGL cells; IL-10 inhibits cytokine synthesis by both T cells and NK cells, but these inhibitory effects are indirect, mediated via the monocyte/macrophage costimulatory cell (1). However, hIL-10 does enhance generation of lymphokine-activated killer activity from resting NK cells in response to IL-2 (7), and also has demonstrable inhibitory effects on subsets of human T cell clones (48). In addition, mIL-10 costimulates growth of mouse thymocytes and T cells (14), and enhances generation of cytotoxic T cell activity (15). NK/LGL cells express a significant level of hIL-10R mRNA, especially when compared to the xcex2-actin reference standard (FIG. 23B). T cells and T cell clones also express readily detectable hIL-10R mRNA, which is apparently down-regulated in the latter in response to activation by anti-CD3 and TPA (FIGS. 23A,C). We do not at present know the significance of the observed down regulation of hIL-10R mRNA expression, especially since it is unclear whether even an 8-10-fold lower hIL-10R number would significantly impair the ability of activated T cells to respond to IL-10. In any case, the expression of significant levels of hIL-10R mRNA by these cells and its regulation by activation stimuli suggest possible additional, as yet uncharacterized effects of IL-10 on T cells and NK cells.
That the Kd of hIL-10R is larger than that obtained for 125I-hIL-10 binding to mIL-10R (xcx9c70 pM) expressed on transfected Ba/F3 (BaMR29) cells (23) is consistent with the observation that hIL-10 has a reproducibly higher specific activity in stimulation of BaMR29 cells compared to Ba8.1 cells (AH and KWM, unpublished). This finding may suggest an intrinsically higher affinity of mIL-10R for ligand. Alternatively, it is possible that additional receptor or signalling components provided in trans by (murine) Ba/F3 cells interact much more efficiently with mIL-10R than with hIL-10R due to the species difference.
Like the cellular cytokine, vIL-10 inhibits macrophage activation and sitimulates human and mouse B cells (1). Thus, further evidence for the possible existence of additional IL-10R components on human B cells and activated monocytes was the finding that vIL-10 at 100-fold molar excess did not detectably compete with FLAG-hIL-10 for binding to hIL-10Rxe2x80x94although at 500-fold molar excess, vIL-10 competed slightly with FLAG-hIL-10 (data not shown). Moreover, we have so far been unable to detect significant levels of binding of FLAG-vIL-10 to either Ba8.1 or COS7 cells expressing hIL-10R using FACS analysis.
hIL-10R, along with mIL-10R (23), are new members of the class II subgroup of CR, the IFNR family. Many of the activating effects of IFNxcex3 on macrophages are inhibited by IL-10 (1). The structures of the ligand-binding chains of IFNxcex1xcex2R and IFNxcex3R are known (43, 50, 51), but biological and genetic evidence has been accumulated for the existence of an additional IFNR polypeptide(s) involved in transduction of a biological signal (see for example (52)). In this light, and in view of the demonstration of shared subunits among receptors for different cytokines (53, 54), it is possible that IL-10R could likewise share a second receptor chain with an IFNR. If so, IL-10, and IFNy might compete for binding to each other""s receptors, but as noted above, neither IFNAxcex1 nor IFNxcex3 competed for hIL-10 binding to recombinant hIL-10R. However, in the IL3, IL5, and GMCSF receptor system (45) a group of cytokine receptors shares a common secondary chain, but each CR is only capable of binding its respective cytokine. A further possibility is that the IL-10-IL-10R interaction might directly antagonize the IFNR signal transduction pathway (55, 56), perhaps by interacting with or sequestering one or more of its components. Further characterization of the structure and signal transduction mechanisms of IL-10R and IFNR may illuminate these possibilities.
Acknowledgments
The authors thank Dr. T. Kitamura for his continuing interest in this work and for valuable advice; Drs. J. Gabert and T. Kitamura for samples of TF-1 mRNA; S. Zurawski for teaching us receptor binding assay techniques; Dr. M. Billah for information and advice regarding DMSO induction of U937 cells; Drs. C.-C. Chou and J. Tan for sharing information before publication; Dr. D.-H. Hsu for contributions to the early phase of this project; J. Polakoff, J. Cupp, and E. Callas for assistance in cell sorting and analysis; and D. Robison for synthetic oligonucleotides. A. S.-Y. Ho was supported by a DNAX Research Institute postdoctoral fellowship. DNAX Research Institute is supported by Schering-Plough Corporation.
1. The sequence reported herein has been deposited in the Genbank database, Accession No. U00672
Abbreviations
CR, cytokine receptor; CSIF, cytokine synthesis inhibitory factor; ELISA, enzyme-linked immunosorbent assay; FACS, fluorescence-activated cell sorter; IFN, interferon; IL, interleukin; LGL, large granular lymphocyte; ORF, open reading frame; PBMC, peripheral blood mononuclear cells; PCR, polymerase chain reaction; SA-PE, streptavidin-phycoerythrin conjugate